1. Field of the Invention
The presently disclosed invention relates to the analysis of bilirubin in a test sample. More particularly, it relates to an improved test kit of the test mat/reagent tablet format for use in bilirubin determination.
The chemistry and biology of the bile pigments are quite complicated; some of the steps in the metabolic pathways being still shrouded in mystery. Much of the older literature on the subject is now obsolete, and not of primary concern to the clinical chemist. Bilirubin, however, is one of the bile pigments occurring somewhat early in the metabolism of heme, and substantial analytical literature is available.
Bilirubin originates primarily from the breakdown of the heme moiety of hemoglobin in senescent erythrocytes by the reticulo-endothelial system. This occurs primarily in the spleen, liver and bone marrow.
Bilirubin which is formed from the breakdown of hemoglobin is transported in the plasma bound to a carrier such as albumin or, in small amounts, .alpha.-globulins and other serum proteins. Anionic drugs, such as salicylates and sulfa, or other anions, such as free fatty acids, can compete for these binding sites and substantially reduce the bilirubin transport capacity of the plasma. It is hypothesized that bilirubin dissociates from its carrier protein in the liver cell membrane, and it is transported intracellularly by some act or process, either unbound or attached to high-affinity, specific acceptor systems.
Conjugation of bilirubin with glucuronic acid and, to a lesser extent with sulfuric and possibly other acids, occurs in the liver. Conjugated bilirubin is excreted from the liver cell into the bile canaliculus. In the intestinal tract a small fraction of the conjugated bilirubin excreted in the bile is hydrolyzed and the unconjugated bilirubin reabsorbed. Consequently, practically all excreted bilirubin is in the conjugated form.
The diagnostic value of determining bilirubin in urine has long been recognized. Normal urinary bilirubin levels are less than about 0.05 milligrams per deciliter (mg%). Accordingly, elevated bilirubin levels in urine connote the possible existence of a pathological condition in a patient. For example, high bilirubin levels can result from biliary obstruction, and hemolytic and hepatic disease. It is generally recognized that the presence of bilirubin in urine at concentrations greater than the above mentioned 0.05 mg% indicates the desirability of performing more comprehensive diagnostic procedures determinative of the specific pathological condition causing the elevated urinary bilirubin concentration.
As stated supra, essentially all bilirubin appearing in pathological urines or other bodily excreta is in the glucuronate conjugated form. Many analytical systems exist in the art for determining this form of bilirubin.
The preparation and use of a bilirubin test device is described in detail in U.S. Pat. No. 3,585,001. While the test devices which have been described in the art provide very rapid and convenient means for detecting urinary bilirubin, it is generally known that the available test devices are not sufficiently sensitive to detect levels of bilirubin only slightly elevated from the normal level, i.e., between 0.1 and 0.4 mg% bilirubin.
There have been a few reported attempts to increase the sensitivity of the reaction between diazonium compounds and urinary bilirubin; however, the test systems that have resulted have certain disadvantages. U.S. Pat. No. 3,880,588 describes a class of diazonium compounds designed to enhance the colorimetric response of the azobilirubin complex and to decrease interfering color reactions with urobilinogen, which is structurally and chemically very similar to bilirubin. The described diazonium compounds, unlike the conventional compounds, form interfering colored products with such constituents of urine as homogentisic acid and 5-hydroxyindole-3-acetic acid. The latter is a normal constituent of urine and as little as 1 mg% of such constituent in urine causes false positive results using the diazonium compounds described in this patent.
Another attempt to increase the sensitivity of the test device-incorporated diazonium reagents is described in U.S. Pat. No. 3,853,476 which discloses the use of certain phosphoric acid diesters as sensitizing or potentiating agents for the reaction between the diazonium compound and bilirubin. However, due to the incompatibility between the phosphoric acid diesters and aqueous media, test devices prepared according to this patent must be manufactured by a double-impregnation process.
It should be mentioned that various so-called "accelerating agents" have been described in the art relative to the detection of bilirubin in serum by the diazo-coupling reaction. Such agents have included caffeine, dyphylline, sodium acetate, sodium benzoate, gum arabic, and various other chemically unrelated compounds.
2. Description of the Prior Art
Over 70 tests have been proposed for the qualitative determination of bilirubin in urine. In general, these can be grouped into four categories depending on the principle used: (a) observation of the color of the urine sample; (b) the titration of the urine sample with a dye (e.g., methylene blue) until the dye color dominates over the bilirubin color; (c) oxidation of bilirubin to characteristic colors; and (d) diazo-coupling. Most of those tests which are clinically feasible rely on diazo-coupling.
Bilirubin, in the presence of certain diazonium compounds, cleaves at its central methylene group to form two molecules of pigment known as azobilirubin. These molecules appear blue to purple at acid and basic pH and red at neutral pH. The reaction can be visualized structurally as follows: ##STR1## where Me is methyl and V is vinyl.
In 1953, Free and Free introduced a tablet test utilizing the diazo-coupling technique in surprisingly convenient form. This test is available commerically as ICTOTEST.RTM. by the Ames Division of Miles Laboratories, Inc. (See Free et al., Gastroenterology, 24, 414 (1953); and U.S. Pat. No. 2,854,317, issued to Free et al. on Sept. 30, 1958.)
ICTOTEST utilizes a test mat capable of absorbing an aqueous test sample, while adsorbing bilirubin which is present, thereby concentrating the analyte. Subsequent to applying several drops of test sample to a spot on the test mat, a reagent tablet containing a stabilized, buffered diazonium reagent composition is placed over the wetted area of the test mat. The tablet is then "flooded" with a few drops of water, thereby partially dissolving the reagents and contacting any adsorbed bilirubin with the diazonium salt-laden solution. If the test sample contains bilirubin a purple color develops in the test mat. This test has been found to be sensitive at bilirubin concentrations as low as 0.05 to 0.10 mg%.
It is to improving ICTOTEST that the present invention relates, and in particular, to improving the test mat. Free et al. teach the use of an asbestos-containing cellulose as the preferred composition for a test mat. Asbestos has the property of being especially suited to adsorbing bilirubin, while absorbing aqueous test samples. However, reference to "The Merck Index", 9th ed., P.110, Merck & Co., Inc. (1976), reveals that asbestos has associated with it potentially serious health hazards. Moreover, alternatives to asbestos suggested by Free et al., which includes Celite (silicon dioxide) and clays have associated with them their own attendant potential health hazards while not possessing sufficient aqueous absorbance coupled with bilirubin adsorbance.
Accordingly, the present invention is the culmination of an attempt to find materials for use in formulating a test mat which, while capable of adsorbing bilirubin to a high degree and otherwise fulfilling the requirements of the ICTOTEST test mat, nevertheless do not embody the hazardous drawbacks attendant asbestos and silicon dioxide. It is this twofold requirement of a test mat--a high degree of absorbance for an aqueous test sample, thereby permitting relatively large sample inoculation at a single point on the mat; coupled with a likewise high propensity to adsorb bilirubin, thereby concentrating it at the point of inoculation--that renders the invention unique.